Prevalence and assemblage identified of Giardia duodenalis in zoo and farmed Asiatic black bears (Ursus thibetanus) from the Heilongjiang and Fujian Provinces of China

Captive and free-living wildlife serve as significant hosts for Giardia duodenalis. Asiatic black bears, valued for their economic and medicinal importance, are extensively farmed in China and also prevalent in zoos. However, studies on G. duodenalis in these animals in China are limited. Here, 218 feces samples of Asiatic black bears were collected: 36 from a zoo in Heilongjiang Province, and 182 from a farm in Fujian Province. Nested PCR of the SSU rRNA gene, followed by sequencing, was employed to determine the frequency and assemblage distribution of G. duodenalis. Positive samples underwent further analysis through multilocus genotyping (MLG) by amplifying the genes for glutamate dehydrogenase (gdh), β-giardin (bg), and triosephosphate isomerase (tpi). Of the 218 samples, G. duodenalis was detected in 22 cases at the SSU rRNA gene locus, including three from Heilongjiang and 19 from Fujian. Three assemblages were identified: A (n = 1), B (n = 16), and E (n = 2) in Fujian; and B (n = 3) in Heilongjiang. Out of the 22 positive samples, 20, 19, and 9 were effectively amplified and sequenced across the tpi, gdh, and bg loci, respectively. Seven samples were genotyped successfully at all three loci, identifying MLG-B1 (n = 1), MLG-B2 (n = 1), and MLG-B3 (n = 1), MLG-B4 (n = 1), MLG-B5 (n = 2), and MLG-B6 (n = 1) as the six assemblage B MLGs. This study marks the first documentation of G. duodenalis in Asiatic black bears in captivity in Fujian and Heilongjiang. The identification of zoonotic assemblages A and B, along with E, underscores potential public health concerns.


Introduction
Giardia duodenalis, a major cause of parasite-related diarrheal illness around the world, affects people and many animals, such as amphibians, birds, and mammals [6,19].This protozoan parasite leads to around 280 million cases of giardiasis annually in humans, causing diarrhea or other intestinal symptoms, with asymptomatic infections being equally prevalent [8].Recognizing its impact, the World Health Organization (WHO) classified giardiasis among Neglected Diseases in 2004 [21].The cysts of G. duodenalis, the infection stage, are excreted in feces, facilitating its spread through the fecal-oral route [25].These cysts can survive in water and other environments, even in the presence of chlorine disinfectants, which accounts for their role in numerous water-borne disease outbreaks [15].Over the past four decades, at least 132 water-borne outbreaks of giardiasis have been documented [7].Food-borne outbreaks have also occurred, associated with contaminated food handled by infected food handlers, placing G. duodenalis 11th among 24 food-borne parasite species listed by the Food and Agriculture Organization of the United Nations (FAO) [18].Despite the global focus on G. duodenalis due to its capacity to cause outbreaks, treatment options remain limited, with no clinically approved vaccines available [1].Effectively addressing G. duodenalis infections requires a thorough understanding of the sources of infection and transmission dynamics.
The application of molecular diagnostics to investigate G. duodenalis infections presents a significant advancement in understanding the epidemiology of this parasite.Molecular typing tools focusing on specific parasitic genes, such as the small subunit ribosomal RNA (SSU rRNA), glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi), and b-giardin (bg), are crucial for identifying G. duodenalis assemblages [28].Use of these tools provides extensive information regarding the spread and possibility for transmission to humans of G. duodenalis assemblages among different animal hosts.Molecular analyses have categorized G. duodenalis into assemblages A to H, all found in mammals but with distinct host distributions [11].Humans and a variety of other mammals are infected by Assemblages A and B, demonstrating a variety of hosts and pandemic transmission capability [6,19].Conversely, Assemblages such as C, D, E, F, G, and H exhibit greater host specificity and more constrained host ranges, with C and D primarily infecting canines, E found in cloven-hoofed domestic mammals, and F, G, and H prevalent in cats, rodents, and marine pinnipeds, correspondingly [11,16,19].Notably, there have been documented cases of C, D, E, and F assemblage infection in humans, especially among children and immunocompromized individuals, underscoring the zoonotic transmission role in human giardiasis epidemiology [6,9,11,20,22].Understanding the distribution of G. duodenalis assemblages in different hosts is crucial for addressing giardia outbreaks effectively.
Illnesses associated with G. duodenalis have increasingly been verified in both captive and free-ranging wildlife through molecular techniques [19].Unlike Cryptosporidium, which shows in species that are host-adapted and genotypes predominating in wild animals, zoonotic G. duodenalis Assemblages A and B are frequently identified in numerous studies, indicating that animals in the wild may act as significant reservoirs for human illnesses [11,19,20].Despite this, genetic proof directly associating G. duodenalis in wild animals with human giardiasis remains limited.Outdoor enthusiasts and caretakers of captive wildlife, who may come into contact with animal excreta, face potential transmission risks from wildlife to humans [19].Bears, including grizzly bears in Canada, Sun bears and brown bears in Croatia, brown bears, American black bears and Andean bears in Peru, and polar bears in Arctic Alaska, have all been reported to carry G. duodenalis [3,16,17,26].This evidence suggests that bears could play a crucial part in the spread of G. duodenalis and warrant attention.Therefore, routine monitoring and genetic recognition of Giardia species/assemblages in these animals are critical for elucidating transmission dynamics and assessing the role of wildlife reservoirs in spreading infections to humans.
In China, an estimated 28.5 million cases of giardiasis occur annually, most of which remain unreported [10].Recent findings have identified G. duodenalis in various animals, emphasizing farm animals (pigs, sheep, and cattle) and pets (dogs and cats) [20].Asiatic black bears, valued for their economic and medicinal importance, are extensively farmed and also prevalent in zoos for commercial and esthetic purposes.As of now, there are no documented known G. duodenalis infections in bears within China.This study investigates the incidence and assemblage dissemination of G. duodenalis in bears from Heilongjiang and Fujian Provinces, using multilocus genotyping (MLG) analysis of the SSU-rRNA, tpi, gdh, and bg genes.The objective is to expand understanding of G. duodenalis occurrence, prevalence, and assemblage distribution in bear populations, thereby enhancing public health knowledge and aiding in the development of preventive strategies.

Ethical declaration
The Wenzhou Medical University Research Ethics Committee and the Animal Ethics Committee gave approval for the study design.The owners or managers of the animals gave their express permission for fecal samples to be taken, guaranteeing that no animals suffered injury or discomfort while the samples were being taken.

Collection of fecal specimens
About 50 g of fresh feces were removed from each of the 218 Asiatic black bears between May 2015 and December 2017.Of these bears, 36 were in a zoo in Heilongjiang Province, and 182 were on a farm in Fujian Province, China (Table 1).Fresh feces samples were gathered from the ground immediately after excretion utilizing sterilized disposable latex gloves, stored in marked sterile bags, and taken to the lab.The samples were kept at 4 °C and processed within 12 h of being collected.

Data logs
Data on the Asiatic black bears, comprising sex, age, and fecal characteristics, were recorded by the trainers and organized into a database using MS Excel 2007.In Fujian Province,

Extraction of DNA
Feces samples were centrifuged at 1500Âg for 10 min after passing using an 8.0-cm-diameter strainer with 45 lm pores.Following the supplier's instructions, genomic DNA was subsequently extracted from 200 mg of the concentrated feces sample through a QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany).The isolated DNA was kept cold for use in PCR testing later on.

Amplification by PCR
To determine whether G. duodenalis was present in the DNA samples, nested PCR was used to target the SSU rRNA gene.The bg, tpi, and gdh genes were the main focus of MLG analysis performed on samples that tested positive for the SSU rRNA gene by nested PCR.The primers and PCR reaction conditions for amplifying the SSU rRNA (~290 bp), tpi (~530 bp), bg (~510 bp), and gdh (~530 bp) genes followed the methodologies illustrated by Appelbee et al., Lalle et al., Sulaiman et al.,and Cacciò et al.,respectively [2,5,13,23].All PCR amplifications used TaKaRa Taq DNA Polymerase (TaKaRa Bio Inc., Tokyo, Japan).DNA from a brown ratderived G. duodenalis assemblage G acted as positive controls in assays of PCR for the SSU rRNA, bg, gdh, and tpi genes to ensure effective amplification and verify DNase absence.DNase-free water served as a negative control in each assay of PCR to monitor for contamination.The 1.5% agarose gel electrophoresis of the PCR results was followed by a Gel Doc EZ UV-gel imaging system (Bio-Rad Inc., Hercules, CA, USA) for graphical representation, which was further improved by a GelRed (Biotium Inc., Hayward, CA, USA) staining process.

Analysis and sequencing of nucleotides
PCR amplicons generated from positive nested PCR reactions targeting G. duodenalis were directed to Sangon Biotech Co., Ltd.(Shanghai, China) for purification and sequencing.The sequencing utilized a Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA) on an ABI PRISM 3730 XL DNA Analyzer.To ensure sequence accuracy, bidirectional sequencing was performed, with additional PCR products sequenced as required.The sequences were edited using DNASTAR Lasergene EditSeq v7.1.0(http://www.dnastar.com/),and Clustal X v2.1 (http:// www.clustal.org/)was used to align the altered sequences with reference sequences that were downloaded from GenBank.

Examining data statistically
The prevalence of G. duodenalis and its association with risk factors such as geographical location, age, gender, and feeding mode were analyzed using the chi-square (v 2 ) test in SPSS software version 22.0 (SPSS Inc., Chicago, IL, USA).Statistical significance was determined at a p-value threshold of less than 0.05.

Nucleotide sequence accession numbers
The nucleotide sequences of G. duodenalis obtained in the present study were deposited in the GenBank database under accession numbers PP356052 to PP356057 for the SSU-rRNA gene, PP388936 to PP388938 for the tpi gene, PP388939 to PP388942 for the gdh gene, and PP388943 to PP388948 for the bg gene.

Molecular characterization
Identification of Giardia assemblages Three assemblages (A, B, and E) of G. duodenalis were found in the bears using sequencing of the SSU rRNA gene.Assemblage B was predominant, representing 86.4% (19/22) of the cases, and was found in animals from the provinces of Fujian and Heilongjiang.Isolates belonging to assemblage A (4.5%, 1/22) and assemblage E (9.0%, 2/22) were exclusively found in bears from Fujian Province.Regarding age distribution, assemblage B was present across all age groups, while assemblages A and E were noticed solely in animals aged 3-5 years.Assemblages A and E were recognized only in solitary male bears.In contrast, assemblage B was observed in bears from both solitary and group feeding modes and in both genders (Table 1).
Genetic diversity of Giardia assemblage at the SSU-rRNA locus Among the 19 SSU rRNA sequences of assemblage B, four haplotypes were recognized, labeled ssuB1 to ssuB4.The ssuB1 haplotype (n = 13) matched 100% with a previously identified G. duodenalis assemblage B sequence (HQ616612) from Lemur catta in Spain, alongside 91 other sequences in GenBank.The remaining six SSU rRNA sequences of assemblage B were novel, showing only minor variations from the ssub1 sequence.Specifically, ssuB2 (n = 4), ssuB3 (n = 1), and ssuB4 (n = 1) sequences each had a single nucleotide difference at positions 6 (C to T), 7 (A to G), and 138 (G to A) relative to ssuB1, respectively.The SSU sequence of assemblage A matched a sequence that had been reported from Lynx pardinus from Spain and another 48 sequences stored in GenBank.Both SSU sequences of assemblage E were previously described, demonstrating homology to a G. duodenalis assemblage E sequence discovered in sheep in China and another 15 sequences stored in GenBank (Table 2).

Genotyping at the triose phosphate isomerase locus
At the tpi locus, sequencing successfully analyzed only 9 of the 22 isolates (40.9%), with none previously described.Seven of these sequences were identical (tpiB) and shared 99.62% similarity with the assemblage B sequences MG736281 and KX085491 from humans in Egypt and Brazil, respectively, with nucleotide substitutions at positions 12 (C to T; G to T) and 490/24 (C to T; A to G).The remaining two sequences, designated tpiE1 and tpiE2, exhibited 99.81% similarity to the assemblage E sequence MH079446 from cattle in China, varying between positions 138 (G to A) and 101 (C to T), accordingly, by a single nucleotide (Table 2).

Genotyping at the beta-giardin locus
Sequencing successfully analyzed 20 of the 22 samples (90.9%) at the bg locus, but no sequences were obtained for the two assemblage E samples.BLAST analysis identified six haplotypes, designated bgB1 to bgB5 for Assemblage B and bgA for Assemblage A. Haplotypes bgB1 (n = 11) and bgB2 were identical to subassemblage BIII isolates (OM115991 and OM115989) from humans in Iran.Haplotypes bgB3 to bgB5 each displayed a single base deletion compared to sequences KJ888976 from Macaca mulatta in China, MT487587 from a masked palm civet in China, and MG736242 from a human in Egypt, respectively.The bgA haplotype showed 99.57% similarity to sequence ON168867 from a pig in China, with two base transitions at positions 18 (A to G) and 366 (G to A) (Table 2).

Glutamate dehydrogenase locus genotyping
Clear amplification products were derived from 19 of the 22 isolates (86.4%) at the gdh locus using generic primers.Four haplotypes, gdhB1 to gdhB4, were identified within Assemblage B. The gdhB1 sequence, representing eight samples, matched 100% with the sequence EU834844 from a human in Belgium.Haplotypes gdhB2 to gdhB4 are novel, with nucleotide transitions observed between gdhB1 and gdhB2 at loci 104 (T to C) and 365 (G to A), between gdhB1 and gdhB3 at sites 104 (T to C), 146 (T to C), and 299 (T to C), and between gdhB1 and gdhB4 at sites 104 (T to C), 146 (T to C), 173 (T to C), 299 (T to C), 308 (C to T), and 463 (A to G).No sequences were obtained for the three samples belonging to Assemblage A or E at the gdh locus (Table 2).

Discussion
This study represents, as far as we are aware, the initial documentation of G. duodenalis in Chinese bears uncovering  [16].A direct comparison with other regions is challenging due to the limited comprehensive data from previous studies.Several factors, including the sample size, examination methods, immune status of the animals, and seasonal variations, influence the infection rates within specific hosts.Notably, bears younger than 5 years exhibited significantly higher infection rates than those older than 5 years, suggesting increased susceptibility to G. duodenalis infection among younger bears.These findings are consistent with research conducted on other animal species, such as cattle, cats, and dogs [4,24].An exhaustive examination and systematic review of stool sample prevalence studies revealed that G. duodenalis is more prevalent between young dogs and cats, with similar observations noted in calves within the cattle population [4,24].This study found a lower prevalence of G. duodenalis in female bears than in male bears, although the relationship between gender and infection rate remains unclear.While gender may not directly influence G. duodenalis infection rates, offspring of infected mothers, particularly during breastfeeding, have a higher likelihood of transmission, highlighting increased susceptibility among children due to close maternal contact.Feeding methods also influence the infection rate, with group-fed animals exhibiting a G. duodenalis prevalence of 17.8%, 3.6 times higher than the 4.9% observed in individually-fed animals.This disparity is attributed to the greater interaction among animals during group feeding, facilitating the spread of infection, thereby underlining the importance of hygiene practices in controlling G. duodenalis infections.Geographical variations in prevalence were observed, with animals in Fujian showing higher rates than those from Heilongjiang, although differences were not statistically significant.The smaller sample size from Heilongjiang than Fujian may have affected the observed prevalence variations.Hence, larger-scale surveys are recommended to explore the relationship between G. duodenalis prevalence and related risk variables further.Analysis across four loci (SSU rRNA, gdh, bg, and tpi) confirmed the existence of three G. duodenalis assemblages (A, B, and E) in the black bears examined.Assemblage B was significantly prevalent, aligning with findings in Sun bears and brown bears in Croatia, where this assemblage predominates [16,26].This genotype is prevalent in humans and various animal species in China, including non-human primates, pigs, cattle, sheep, horses, and other wild animals [14,27].Assemblage A is also common in China among both humans and non-human primates as well as pigs, livestock, and sheep, and was detected in one bear, indicating a potentially broader host range [14,27].The existence of assemblages A and B underscores the potential of bears as reservoirs for human infections, highlighting the need for further research on interspecies transmission.
In the present research, assemblage E of G. duodenalis was detected alongside assemblages A and B. Assemblage E, predominantly found in livestock and previously regarded as ruminant-specific, has evidence suggesting its zoonotic transmission potential between humans and animals [11].It has been identified in 54 human cases globally, mainly in rural areas, with individuals presenting gastrointestinal symptoms, with or without diarrhea [10].Molecular analyses have revealed identical genetic types in human cases and livestock (cattle and sheep), indicating a strong likelihood of zoonotic transmission of Assemblage E [9][10][11]30].The pathway for Assemblage E's introduction to bears remains unclear, although water or food contamination by domestic animals is the most probable route.While Assemblage E is uncommon in wild animals, cross-transmission between wild and domestic animals has been documented, such as in red colobus monkeys from Uganda, suggesting cross-transmission from cattle [12].
The SSU rRNA locus exhibited the highest amplification rate and was the most conserved among the four studied loci (SSU rRNA, tpi, gdh, and bg).In contrast, the tpi locus showed the lowest amplification rate, with positive samples accounting for only 40.9% (9/22).The gdh and bg loci demonstrated high amplification rates and significant genetic variation.This study indicates that the amplification success of the bg, gdh, and tpi genes correlates with the assemblage type, with Assemblages A and B more frequently amplified at the bg and gdh genes, whereas Assemblage E showed greater success at the tpi locus.Similar observations regarding differences in amplification rates across gene loci have been reported in previous research.For instance, Xu et al. investigated the bg, gdh, and tpi genes and yielded 40, 34, and 59 sequences from 129 G. duodenalispositive isolates, respectively, in donkeys in Xinjiang, China [29].Out of 195 positive samples, only 11 and 6 samples, accordingly, were found to have G. duodenalis at the tpi and gdh loci in research conducted in pigs in Fujian Province, China [31].The observed low amplification rates for the gdh, bg, and tpi loci highlight the need for more sensitive typing techniques to enable comprehensive genetic characterization of these isolates.
MLG analysis on three genes identified six novel MLGs (MLG-B1, MLG-B2, MLG-B3, MLG-B4, MLG-B5, and MLG-B6) within assemblage B, indicating the potential existence of unique subassemblages B in G. duodenalis found in bears.Although no MLGs for assemblages A and E were determined due to unsuccessful amplification at all four loci, genetic diversity was evident, as indicated by novel sequences bgA, tpiE1, and tpiE2.This finding underscores the need for further investigation to elucidate the specific mechanisms involved, given the absence of descriptions on MLG typing of G. duodenalis in bears.Future studies are needed to verify the presence of assemblage A and E infections in bears.

Conclusions
This report provides an initial examination of the occurrence of G. duodenalis in black bears farmed and housed in zoos across two provinces in China, detailing the features of the assemblages and assessing the potential for zoonotic transmission.The characteristics of assemblages A, B, and E in bears suggest the capability of these animals to transmit giardiasis to humans and domestic animals.Moreover, detecting novel sequences highlights the possibility of unique regional or

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Chen et al.: Parasite 2024, 31, 50 128 bears were divided into three categories: 17 were under 3 years old, 122 were aged between 3 and 5 years, and 43 were older than 5 years.The gender distribution included 118 males and 64 females, with 92 bears raised individually and 90 in groups.There were 12 bears under 3 years old and 24 bears over 5 years old in Heilongjiang Province.In Heilongjiang, each of the 36 bears was reared separately, comprising 15 males and 21 females.When the samples were being collected, no signs of illness were observed in any animal.

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Chen et al.: Parasite 2024, 31, 50 host-specific correlations, underscoring the need for further investigation to fully comprehend the zoonotic potency and spread dynamics of G. duodenalis within bear populations.

Table 1 .
Prevalence and assemblage of G. duodenalis at the SSU rRNA, bg, gdh and tpi genes in bears according to province, age, gender, and feeding mode.

Table 2 .
Similarity analysis of SSU-rRNA, bg, gdh, and tpi sequences within G. duodenalis assemblage A, assemblage B, and assemblage E.
a Another 91 sequences stored in GenBank.b Another 48 sequences stored in GenBank.c Another 15 sequences stored in GenBank.

Table 3 .
Multilocus characterization of G. duodenalis isolates based on the SSU rRNA, bg, gdh, and tpi genes. .Chen et al.: Parasite 2024, 31, 50 an overall prevalence of 10.1%, with 10.4% in a farm in Fujian and 8.3% in a zoo in Heilongjiang.While reports of Ursidae infections with G. duodenalis exist, only one comprehensive survey from Croatia, revealing an infection rate of 4.3%, has been published on bears J